Key in understanding the ability of muscles to shorten and make force is understanding the interaction of myosin crossbridges with the actin filament. We have been involved in characterizing the behavior of myosin crossbridges when they have ATP bound. During FY '88 we substantiated that at ionic strengths ranging from very low values up to physiological values, measuring the muscle fiber's rapid resting stiffness provides an accurate way of monitoring the interaction of these ATP crossbridges with actin. It was furthermore shown that the binding of the ATP crossbridge in fibers correlates virtually perfectly with the binding of myosin subfragment-l to actin in solution. Since the solution work is done in the absence of the regulatory proteins troponin and tropomyosin, this suggests that the increased binding of ATP crossbridges at low ionic strength is a direct effect of ionic strength on the crossbridge rate constants, unrelated to any changes in troponin and tropomyosin structure which may also occur. Also in FY '88, we looked for ways of studying the influence of crossbridge density on crossbridge behavior. Our plan was to modify just some of the crossbridges in a way that would render them incapable of binding to actin. We tried treating fibers with the bifunctional alkylating agent pPDM, which, in solution, greatly weakens actin binding affinity by crosslinking myosin's SHl and SH2 sulfhydryls. In the fiber, however, this had the deleterious side effect of increasing the fiber's resting tension. We discovered that the monofunctional reagent phenylmaleimide could reduce crossbridge affinity without increasing resting tension. This showed that crosslinking of SHl and SH2 is not necessary for reducing crossbridge affinity and may also have provided us with a tool for studying the influence of crossbridge density on crossbridge behavior. Finally in FY '88, we demonstrated that the ability of smooth muscle to maintain force even after myosin light chain phosphorylation, the trigger for force generation, has returned to near baseline values, is not due to protein kinase C phosphorylating the myosin light chains at serine-l.